RU2712157C2 - Creating controlled release profile of drug substance using liposomal composition in aqueous and anhydrous solutions - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: group of inventions refers to pharmaceutical industry, in particular to embodiments of a pharmaceutical composition for inhibiting growth of cancer cells. In one embodiment, the composition includes a component which forms a particle selected from a phospholipid and a mixture of at least one phospholipid and cholesterol; 0.1–10 mM dextran sulphate ion or its pharmaceutically acceptable salt; 100–500 mM ammonium sulphate; amphipathic therapeutic agent or its pharmaceutically acceptable salt. In another embodiment of the composition, the amount of ammonium sulphate is 150–450 mM, and the amphipathic therapeutic agent is a vinca roseacola alkaloid. Also disclosed are: a liposome for delivering an amphipathic therapeutic agent and a method of inhibiting growth of cancer cells by administering an effective amount of said pharmaceutical composition.EFFECT: group of inventions provides optimal therapeutic effectiveness and reduced toxic effect on skin.16 cl, 4 dwg, 11 tbl, 10 ex

Description

Link to related applications

This application claims priority in accordance with the application for the grant of a US patent with serial No. 61/792850, filed March 15, 2013, the full disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising at least one liposome, at least one multivalent counterion donor or a pharmaceutically acceptable salt thereof, at least one monovalent counterion donor, or a pharmaceutically acceptable salt thereof and a therapeutic agent.

BACKGROUND OF THE INVENTION

Liposomes have been widely used as a carrier of various therapeutic agents in vivo. It is desirable that such liposomes have high encapsulation efficiency and a long retention profile (i.e., the drug substance should be released to a minimum extent before reaching the site of application of the action).

NanoVNB® is a liposome vinorelbine that uses liposomes to enhance the retention of vinorelbine until it reaches the site of action. In phase I of the clinical trials of NanoVNB®, its enhanced anticancer effect was indeed detected, but prolonged in vivo retention of vinorelbine also led to increased toxicity.

Thus, there is a need for a liposome composition that can be used to deliver a therapeutic agent with an adjustable retention profile to achieve a balance between optimal anti-cancer effects and minimal side effects. The present invention is directed to solving this problem, as well as other important tasks.

SUMMARY OF THE INVENTION

According to one embodiment, the present invention relates to a pharmaceutical composition comprising at least one liposome, at least one multivalent counterion donor or a pharmaceutically acceptable salt thereof, at least one monovalent counterion donor, or a pharmaceutically acceptable salt thereof, and a therapeutic agent, derivative thereof, or a pharmaceutically acceptable salt. This pharmaceutical composition advantageously provides an adjustable retention profile and an adjustable percentage of encapsulated therapeutic agent.

According to another embodiment, the present invention relates to a pharmaceutical composition comprising at least one liposome, comprising a particle forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; 0.1 mM to 10 mM multivalent counterion donor or a pharmaceutically acceptable salt thereof; from 150 mM to 450 mM of a monovalent counterion donor or a pharmaceutically acceptable salt thereof; and alkaloid vinca pink.

According to a third embodiment, the present invention relates to a pharmaceutical composition comprising at least one liposome, comprising a particle forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; from 1 milliequivalent (meq.) to 320 meq. a polyvalent counterion donor or a pharmaceutically acceptable salt thereof; from 150 mM to 450 mM of a monovalent counterion donor or a pharmaceutically acceptable salt thereof; and amphipathic therapeutic agent.

The present invention also relates to methods for inhibiting cancer cell growth in a subject in need thereof. The method comprises the step of administering the pharmaceutical composition described herein, wherein the subject decreases the severity of the symptoms and signs of the malignant tumor. This method advantageously enhances inhibition of cancer cell growth and reduces toxicity.

Statements containing these terms should be construed so as not to limit the present invention described herein or to limit the meaning or scope of protection of the following patent claims. Embodiments of the present invention that are covered in this patent application are defined by the following claims, and not this brief disclosure. This brief disclosure is a general overview of various aspects of the present invention, and it discusses some concepts that are further described in the following detailed disclosure section of the present invention. This brief disclosure is not intended to establish key or essential features of the claimed invention, nor is it intended to be used separately to determine the scope of protection of the present invention. The subject matter of the invention should be understood with reference to the relevant sections of the full description, any or all of the graphic materials and each claim.

The present invention will become more apparent when considering the text of the application along with the accompanying graphic materials and a detailed description, which are given below.

Brief Description of the Figures

In FIG. 1 shows the precipitation of the sodium salt of dextran sulfate and vinorelbine in a liposome.

In FIG. Figure 2 shows the average tumor volume in the group treated with NanoVNB, in the group treated with LV304, and the group (control) treated with saline.

In FIG. 3 shows the average survival time in the group treated with NanoVNB, in the group treated with LV304, and the group (control) treated with saline.

In FIG. 4 shows an indicator of skin toxicity in the group treated with NanoVNB and in the group treated with LV304.

Detailed disclosure of the present invention

Definitions

It should be understood that the terms above and throughout the disclosure, unless otherwise indicated, have the following meanings.

As used herein, the singular forms include the reference to the plural unless the context clearly indicates otherwise.

As used herein, the term "effective amount" encompasses a dose of a pharmaceutical composition that is sufficient to reduce the severity of symptoms and signs of a malignant tumor, which include, without limitation, weight loss, pain and tumor mass, which can be detected either clinically as a palpable formation, or using various means of radiation diagnostics.

As used herein, the term “treatment”, “receiving treatment” or “treatment” encompasses preventive (eg, prophylactic), palliative and therapeutic use or results.

The term "inhibition" and "suppression" encompasses the inhibition or prevention of growth.

The term “subject” may mean a vertebrate having a malignant tumor, or a vertebrate that is believed to need treatment for a malignant tumor. Subjects include warm-blooded animals, such as mammals, such as a primate, and more preferably humans. Lower primates also apply to subjects. The term “subject” encompasses domesticated animals such as cats, dogs, etc., farm animals (eg, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (eg, mouse, rabbit , rat, gerbil, guinea pig, etc.). Therefore, this document provides for the use of drugs in veterinary medicine.

The term “counterion donor” encompasses a counterion donor capable of forming a salt with a therapeutic agent and not reducing the activity of the therapeutic agent. In one embodiment, the therapeutic agent is an amphipathic acid with a net negative charge, the counterion donor is a cationic donor or molecule coupled covalently to one or more cationic functional groups. According to another embodiment, the therapeutic agent is an amphipathic base with a total positive charge, the counterion donor is an anion donor or a molecule connected by a covalent bond to one or more anionic functional groups. The counterion donor has high solubility in the component carrying the agent in the liposome, but low permeability through the liposome membrane (bilayer). Thus, the counterion donor is held in the component carrying the agent during the load of the therapeutic agent and during storage.

All numbers in this document can be understood with the term "approximately".

As used herein, the term “alkyl” means a linear or branched, saturated, aliphatic radical having from 1 to about 10 carbon atoms. Alkyl may include any number of carbon atoms, such as, for example, C _1-2 , C _1-3 , C _1-4 , C _1-5 , C _1-6 , C _1-7 , C _1-8 , C _{1- 9} , C _1-10 , C _2-3 , C _2-4 , C _2-5 , C _2-6 , C _3-4 , C _3-5 , C _3-6 , C _4-5 , C _{4- 6} and C _5-6 . For example, C _1-6 alkyl includes, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl may also mean alkyl groups having up to 20 carbon atoms, such as, without limitation, heptyl, octyl, nonyl and decyl.

As used herein, the term “aryl” means an aromatic ring system having any suitable number of atoms in a ring and any suitable number of rings. Aryl groups may include any suitable number of atoms in the ring, such as, for example, 6, 7, 8, 9,10, 11, 12, 13,14, 15 or 16 atoms in the ring, as well as from 6 to 10, from 6 to 12 or 6 to 14 elements of the cycle. Aryl groups can be monocyclic groups of 15 atoms, which condense to form bicyclic or tricyclic groups, or are connected by bonding to form a biaryl group. Typical aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl having a methylene linking group. Some aryl groups have 6 to 12 ring elements, such as phenyl, naphthyl or biphenyl. Other aryl groups have 6 to 10 ring elements, such as phenyl or naphthyl. Some other aryl groups have 6 to 20 ring elements, such as phenyl. "Substituted aryl" groups may be substituted by one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano and alkoxy groups.

“Pharmaceutically acceptable salts” of the amphipathic acid of the present invention are salts formed with bases, namely cationic salts, such as alkali and alkaline earth metal salts such as sodium, lithium, potassium, calcium, magnesium, and also quaternary ammonium salts, such as for example, ammonium, trimethylammonium, diethylammonium and tris (hydroxymethyl) methylammonium salts.

Similar acid addition salts, such as salts of mineral acids, organic carboxylic and organic sulfonic acids, for example, hydrochloric acid, methanesulfonic acid, maleic acid, may also be formulated in the main therapeutic agent with a component such as pyridyl included in the composition structure.

Liposome

As used herein, the term “liposome” means multivesicular liposome (MVL), multilamellar vesicles (MLV), or small or large monolamellar vesicles (ULV). Liposomes are nanoparticles in size and contain a particle-forming component and a substance carrier component. The particle-forming component forms a closed lipid barrier, and the carrier component of the substance is a medium surrounded by a particle-forming component.

The particle forming component can be obtained from a phospholipid or a mixture of at least one phospholipid and cholesterol. Examples of the phospholipid used in the present invention include, but are not limited to, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (RA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidic acid (EPA), egg phosphatidylinositol (EPI), soybean phosphatidylcholine (SPC), soybean phosphatidylglycerol, serine phosphatidylglycerol phosphatidylserine (SPS), soybean phosphorus atonic acid (SPA), soybean phosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphylhydroglyphosphorylphosphorylphosphorylphosphorylphosphate (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylethanolamine (DOPE), palmitoilstearilfosfatidilholin (PSPC), palmitoilstearoilfosfatidilglitserin (PSPG), monooleo lphosphatidylethanolamine (SEA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), distearylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-phosphine-glycerol-d-glycerol , dimyristoylphosphatidylserine (DMPS), distearylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphate acid (DPA), DPA, DPA ), 1,2-dioleyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoylphosphatidylinositol (DMPI), dis thearylphosphatidylinositol (DSPI) and a mixture thereof.

In one embodiment, the particle forming component does not contain a fatty acid or a cationic lipid (i.e., a lipid having a net positive charge at physiological pH).

In another embodiment, the particle forming component comprises a hydrophilic polymer, which is a long chain, highly hydrated, neutral polymer with a flexible structure that is attached to a phospholipid molecule. Not limited to any theory, it is believed that the hydrophilic polymer stabilizes the liposome and increases the half-life in the circulatory bed in vivo. Examples of the hydrophilic polymer include, without limitation, polyethylene glycol (PEG) with a molecular weight of from about 2000 to about 5000 daltons, methoxy-PEG (mPEG), GMi ganglioside, polysialic acid, polylactic acid (also called polylactide), polyglycolic acid (also called polyglycolide ), a copolymer of polylactic and polyglycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyl oxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxy ropilmetakrilamid, polymethacrylamide, polidimetilakrilamid, polivinilmetilefir, poligidroksietilakrilat, derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose, and synthetic polymers.

According to one group of embodiments, the phospholipids are selected from DSPC and DSPE-PEG, wherein the molecular weight of the PEG is approximately 2000 daltons (hereinafter referred to as DSPE-PEG ₂₀₀₀ ).

According to another group of embodiments, the molar ratio of DSPC, cholesterol and DSPE-PEG ₂₀₀₀ is approximately 3: 2: 0.45.

The particle-forming component may further include an antibody lipid conjugate or peptide that acts as an address group to provide specific binding of the liposome to the target cell that contains the target molecule. Examples of target molecules include, but are not limited to, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), cancer-embryonic antigen (CEA), and erbB-2 / neu (HER2).

Liposomes have an average particle diameter of from about 30 nm to about 200 nm, more preferably from about 50 nm to about 150 nm.

The liposomes obtained in the present invention can be prepared using conventional methods used to obtain vesicles. These methods include a method for injecting an ethereal lipid solution (Deamer el al, Acad. Sci. (1978) 308: 250), a method using a surfactant (Brunner elal, Biochim. Biophys. Acta (1976) 455: 322), method freeze-thaw (Pick el al, Arch. Biochim. Biophys. (1981) 212: 186), reverse phase evaporation method (Szoka el al, Biochim. Biophys. Ada. (1980) 601: 559 71), ultrasonic treatment (Huang el al, Biochemistry (1969) 8: 344), method for injecting an ethanolic lipid solution (Kremer el al, Biochemistry (1977) 16: 3932), extrusion method (Hope el al, Biochim. Biophys. Acta (1985) 812: 55 65), a method using a French press (Barenholz etal, FEBSLett. (1979 ) 99: 210) and methods described in detail in Szoka, F., Jr., el al, Ann. Rev. Biophys. Bioeng. 9: 467 (1980). All of the above methods belong to the basic technology for the formation of liposomal vesicles and these methods are incorporated herein by reference.

Therapeutic agent

The therapeutic agent may be any suitable therapeutic agent. In one embodiment, the therapeutic agent is an anti-cancer agent. Non-limiting examples of an anticancer agent include a pink vinca alkaloid, a topoisomerase inhibitor, a taxane compound, derivative thereof, or a pharmaceutically acceptable salt.

Examples of the pink vinca alkaloid include, but are not limited to, vinorelbine, vincristine, vinblastine, and vindestine.

Examples of a topoisomerase inhibitor include, but are not limited to, topotecan, camptothecin, irinotecan, etoposide, and doxorubicin.

Examples of a taxane compound include, but are not limited to, paclitaxel.

Monovalent Counterion Donor

According to one embodiment, the therapeutic agents are amphipathic bases with a total positive charge, the donor of monovalent counterions in the liposome can be selected from an anion or a molecule that is covalently linked to an anionic functional group. Anion or anionic functional group has a valency of -1, -2 or -3.

Non-limiting examples of a monovalent counterion donor include benzenesulfonic acid and 4-hydroxybenzenesulfonic acid, which are illustrated below:

бензолсульфоновая кислота, и

benzenesulfonic acid, and

4-гидроксибензолсульфоновая кислота.

4-hydroxybenzenesulfonic acid.

According to another embodiment, the pharmaceutically acceptable monovalent counterion donor salt comprises a) an anion or molecule that is covalently linked to an anionic functional group; and b) one or more cations, wherein the anion or anionic functional group forms ion pairs with cations.

An anion or anionic functional group can be selected from one or more of the following ions: citrate, sulfate, sulfonate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, carboxylate, glucuronate, chloride, hydroxide, nitrate, cyanate or bromide. In one embodiment, the anion and anionic functional group are selected from one or more of the following ions: citrate, sulfate, sulfonate, phosphate, pyrophosphate, or carboxylate.

In yet another embodiment, the molecule associated with the anionic functional group may be a natural or synthetic, organic or inorganic compound. Examples of such a molecule include, but are not limited to, a molecule of non-polymer nature, such as, for example, benzene, an oligonucleotide and a monosaccharide or a polymer such as polyvinyl, a polyhydric alcohol such as glycerin, sorbitol and mannitol, a polysaccharide, polypeptides, glycoproteins and polynucleotide.

The pharmaceutically acceptable salt cation can be selected from one or more of the following ions: calcium ion, magnesium ion, sodium ion, potassium ion, manganese ion or NR ₄ ⁺ , where R is H or an organic residue, such as alkyl or aryl, or mixture. In one embodiment, the cation is ammonium.

A second embodiment of the present invention relates to an amphipathic therapeutic agent having acidic properties, and the monovalent counterion donor in the liposome can be selected from or includes a cation or molecule that is covalently linked to a cationic functional group. A cation or cationic functional group has a valency of +1, +2, or +3.

A pharmaceutically acceptable monovalent counterion donor salt comprises a) a cation or molecule that is covalently linked to a cationic functional group; and b) one or more anions, the cation or cationic functional group forming ion pairs with one or more anions.

In one embodiment, the monovalent counterion donor is ammonium sulfate. According to another embodiment, the concentration of the monovalent counterion donor is from about 100 to about 500 mM, or includes any value or ranges within these ranges in 10 mM steps (for example, 80 mM, 320 mM). In yet another embodiment, the monovalent counterion donor concentration is from about 50 to about 450 mM. In yet another embodiment, the monovalent counterion donor concentration is from about 200 mM to about 400 mM. In yet another embodiment, the concentration of the monovalent counterion donor is about 300 mM.

Multivalent counterion

In one embodiment, the therapeutic agent is an amphipathic base, and at least one multivalent counterion donor, or a pharmaceutically acceptable salt thereof, forms an insoluble salt in the liposome.

In another embodiment, the multivalent counterion donor comprises a molecule that is covalently linked to several anionic functional groups, the anionic functional group having a valency of -1, -2, or -3. A pharmaceutically acceptable multivalent counterion donor salt includes a) a molecule covalently linked to several anionic functional groups; and b) one or more cations, the anionic functional group forming ion pairs with cations.

The anionic functional group of the multivalent counterion is selected from one or more of the following ions: citrate, sulfate, sulfonate, phosphate, pyrophosphate, tartrate, succinate, maleate, borate, carboxylate, glucuronate, chloride, hydroxide, nitrate, cyanate or bromide. In one embodiment, the anionic functional group is selected from one or more of the following ions: citrate, sulfate, sulfonate, phosphate, pyrophosphate, or carboxylate. Each of the anionic functional groups of the multivalent counterion donor may differ from the other. For example, chondroitin sulfate is a donor of multivalent counterions with different anionic functional groups on the same molecule, as illustrated below:

The cation can be selected from one or more of the following ions: calcium ion, magnesium ion, sodium ion, potassium ion, manganese ion, NR ₄ , where R represents H or an organic residue, such as alkyl or aryl, and mixtures thereof. In one embodiment, the cation is ammonium.

Another embodiment of the present invention relates to an amphipathic therapeutic agent having acidic properties, and the donor of the multivalent counterions in the liposome includes a molecule that is covalently linked to several cationic functional groups, and said cationic group has a valency of +1, +2 or +3. Amphipathic acid forms an insoluble salt with a donor of multivalent counterions and is enclosed in a liposome.

A pharmaceutically acceptable multivalent counterion donor salt comprises a) a molecule that is covalently linked to one or more cationic functional groups; and b) one or more anions, the cationic functional group forming ion pairs with anions.

The multivalent counterion donor molecule may be a molecule of a natural or synthetic, organic or inorganic compound. Non-limiting examples of such a molecule include a non-polymer molecule, such as an oligonucleotide and monosaccharide, or a polymer, such as polyvinyl, polyhydric alcohols, such as glycerin, sorbitol and mannitol, polysaccharides, such as dextran and chitosan, polypeptides, glycoproteins and polynucleotides.

In one embodiment, the multivalent counterion donor is selected from one or more of the following: sulfated heparin, carrageenan, mucin, sulfated hyaluronic acid, chondroitin sulfates, keratin sulfates, dermatan sulfates, or sulfated polysaccharide. A non-limiting example of a sulfated polysaccharide includes dextran sulfate with a molecular weight of from about 1600 daltons to about 8000 daltons.

In one embodiment, the pharmaceutically acceptable dextran sulfate salt is selected from the ammonium salt of dextran sulfate or the sodium salt of dextran sulfate.

Pharmaceutical composition

According to one embodiment, the pharmaceutical composition of the present invention comprises at least one liposome comprising: a particle forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; at least one multivalent counterion donor or a pharmaceutically acceptable salt thereof; at least one monovalent counterion donor or a pharmaceutically acceptable salt thereof; and an amphipathic therapeutic agent, a derivative thereof, or a pharmaceutically acceptable salt thereof.

According to another embodiment, the pharmaceutical composition of the present invention comprises at least one liposome, comprising: a particle forming component selected from a mixture of one or more phospholipids and cholesterol; at least one donor of multivalent counterions or its pharmaceutically acceptable salt with a concentration of from about 0.1 mm to about less than 10 mm; at least one monovalent counterion donor or a pharmaceutically acceptable salt thereof with a concentration of from about 150 mM to about 450 mM; and the vinca pink alkaloid. According to yet another embodiment, the particle forming component further comprises a hydrophilic polymer.

Advantageously, by combining a multivalent counterion donor or its pharmaceutically acceptable salt and a monovalent counterion donor or its pharmaceutically acceptable salt, the encapsulation efficiency and / or the retention profile of a therapeutic agent can be adjusted to provide therapeutic efficacy while minimizing toxicity.

In one group of embodiments, the anionic functional groups of the multivalent counterion donor or its pharmaceutically acceptable salt have a total valency equivalent per liter of from about 1 to about 160 milliequivalents (meq.), From about 3 to 160 meq., From about 1 to about 320 meq. , from about 1 to about 250 meq., from about 3 to about 250 meq., from about 160 to about 250 meq., from about 160 to about 320, or any value whether ranges from 1 to 320 meq. in 1 meq steps (e.g. 23 meq., 233 meq.). According to another embodiment, the anionic functional group of the multivalent counterion donor or a pharmaceutically acceptable salt thereof is a sulfate group.

According to another group of embodiments, the concentration of the multivalent counterion donor or a pharmaceutically acceptable salt thereof is from about 2 mM to less than 8 mM, from about 0.1 mM to less than 8 mM, from about 0.1 mM to less than about 10 mM, from about 2 mm to less than 10 mm or includes any value or ranges in the range from 0.1 mm to 10 mm in 0.1 mm steps (for example, 1.5 mm, 8.3 mm).

The pharmaceutical composition is prepared taking into account any suitable route of administration, including intracranial, intracerebral, intraventricular, intrathecal, intravertebral, oral, local, rectal, intradermal, subcutaneous, intravenous, intramuscular, intranasal, intraperitoneal, intra-tumor, and the like.

The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art according to options for implementation. Dosage forms in the form of single or multiple doses are provided, each of which has advantages in certain clinical situations. According to the present invention, the actual amount of the pharmaceutical composition to be administered can be changed in accordance with the age, weight, general health of the subject to be treated, the type of cancer, toxicity, and depends on the discretion of the medical staff.

In some embodiments, at least a portion of the therapeutic agent (such as vinorelbine) forms a salt with a pharmaceutically acceptable multivalent counterion donor salt and precipitates in the intraliposomal aqueous core, as can be seen from FIG. 1.

Method for Inhibiting Malignant Tumor Cell Growth The present invention relates to methods for inhibiting malignant tumor cell growth in a subject, which comprises the step of administering an effective amount of the pharmaceutical composition described herein to a subject in need thereof, thereby reducing the severity of symptoms and signs in the subject. malignant tumor and / or reduced toxicity.

The pharmaceutical composition can be administered alone or as an adjuvant for surgical intervention, for example, before surgery to reduce the size of the tumor and / or after surgery to reduce the likelihood of metastases, for example, by inhibiting the growth and migration of circulating tumor cells through the bloodstream.

The pharmaceutical composition may be administered before, after, or simultaneously with one or more anti-cancer agents. The anticancer agent includes a conventional chemotherapeutic agent, targeted cancer therapy, or radiotherapy.

A conventional chemotherapeutic agent includes a DNA synthesis inhibitor, an alkylating agent, an anti-folate agent, a metabolic inhibitor, or a combination thereof.

Targeted cancer therapy involves the use of drugs that inhibit the growth of cancer cells by targeting specific target molecules involved in carcinogenesis and growth of the cancer, as opposed to simply acting on rapidly dividing cells (for example, as is the case with conventional chemotherapeutic means). Targeted cancer therapy includes the use of a kinase inhibitor, an angiogenesis inhibitor, an epidermal growth factor receptor inhibitor (EGFR), an HER2 / neu receptor inhibitor, or a combination thereof.

In radiotherapy, high-energy radiation is used to reduce the tumor and destroy the cells of the malignant tumor. Examples of radiotherapy include x-rays, gamma radiation, and charged particles.

The following examples further illustrate the present invention. These examples are provided merely to illustrate the present invention and should not be construed as limiting the scope of protection of the present invention.

Example 1: liposome preparation

Liposomes were prepared by the method of injecting a lipid solution. Lipids, including DSPC, DSPE-PEG ₂₀₀₀ and cholesterol, were combined in a molar ratio of 3: 0.045: 2 and dissolved in 99.9% ethanol at approximately 60 ° C in a vial. To dissolve the lipids used a bench-top ultrasonic bath.

The dissolved lipid solution was added to a 1.0 mM sodium phosphate solution at 100 ml / min using a peristaltic pump, and the two solutions were mixed. Then the lipid mixture was passed 6-10 times through a polycarbonate membrane with pore sizes of 0.2 μm and 0.1 μm, respectively. Liposomes (or large multilamellar vesicles) formed, and the average diameter of the vesicles was approximately 100-120 nm (measured using the Malvern ZetaSizer Nano ZS - 90).

The liposome mixture was dialyzed and concentrated using a tangential flow filtration system against 0.9% (w / w) sodium chloride and 9% (w / w) sucrose solution using Millipore Pellicon 2 Mini Ultrafiltration Module Biomax-100С (0, 1 m), and then sterilized using a 0.2 μm sterile filter.

Example 2: Effect of a Monovalent Counterion Donor on Encapsulation Efficiency and Retention Profile

A pharmaceutical composition was prepared by mixing the liposomes obtained in Example 1 with ammonium sulfate, a monovalent counterion donor. A gradient was established across the liposome lipid bilayer membrane using 300 mM and 600 mM ammonium sulfate for the remote loading of vinorelbine. The encapsulation efficiency (load) and the retention profile of liposomal vinorelbine were evaluated in vitro using a plasma-based release method, and the results are summarized in Table 1.

Results: data show that ammonium sulfate contributed to the loading or encapsulation of vinorelbine into the liposome. However, ammonium sulfate to a lesser extent contributed to the retention of vinorelbine in the liposome, with less than 30% of vinorelbine remaining encapsulated in the liposome 24 hours after incubation with blood plasma.

Table 1 Characteristics of Pharmaceutical Compositions Containing Monovalent Counterion Donors Pharmaceutical composition Monovalent Counterion Donor Particle Size [nm] Encapsulation Efficiency [%] % of encapsulated vinorelbine remaining 24 hours after incubation with blood plasma LV001 300 mM ammonium sulfate 93.3 93 28.7 LV006 600 mM ammonium sulfate 93.6 93 24.7

Example 3: Effect of a Multivalent Counterion Donor on Encapsulation Efficiency and Retention Profile

From the sodium salt of dextran sulfate with a molecular weight of 8000 (8K) Dalton, an ammonium salt of dextran sulfate (a pharmaceutically acceptable salt of dextran sulfate) was obtained using a DOWEX ion exchange column. Two pharmaceutical compositions were prepared by mixing the liposomes obtained in Example 1 with 4 mM and 8 mM ammonium salt of dextran sulfate, respectively, followed by a distance load of approximately 2 mg of Vinorelbine incubated at approximately 60 ° C.

The encapsulation efficiency and retention profile of liposomal vinorelbine in these two pharmaceutical compositions were evaluated in vitro using a plasma-based release method, and the results are summarized in Table 2.

Results: for 8 mM ammonium salt of dextran sulfate, the encapsulation efficiency was 93%, while the encapsulation efficiency for 4 mm ammonium salt of dextran was below 90%. Similarly, the composition of LV009, shown in Table 4, included only a multivalent counterion donor and was characterized by an encapsulation efficiency of less than 90%, and a retention rate of 98.74% 24 hours after incubation in blood plasma.

table 2 Characteristics of pharmaceutical compositions containing multivalent counterion donors. Pharmaceutical composition Multivalent Counterion Donor Salt Particle size Encapsulation Efficiency Ammonium salt of dextran sulfate (MW = 8K) (nm) (%) LV702 4 mm 114 84 LV703 8 mm 114 93

Example 4: Effect of a combination of donors of mono- and multivalent counterions To study the effect of a combination of donors of mono- and multivalent counterions on the retention profile of liposomal vinorelbine, an in vitro study was performed.

The liposomes prepared according to Example 1 were mixed with 300 mM ammonium sulfate (a donor of monovalent counterions) and various concentrations of the sodium salt of dextran sulfate (a salt of a donor of multivalent counterions).

The encapsulation efficiency and retention profile for various types of liposomal vinorelbine were evaluated in vitro using a plasma-based release method after 24 hours, and the results are summarized in Table 3.

Results: the data demonstrate that various combinations of donors of monovalent and multivalent counterions can maintain the efficiency of encapsulation of vinorelbine, while the size of the liposomes was approximately 100 nm. In addition, the retention profile of liposomal vinorelbine depends on the concentration of the multivalent counterion donor. The use of 8 mM dextran sulfate sodium salt is associated with a higher percent retention of vinorelbine after 24 hours (78.9%) than the use of 2 mM dextran sulfate sodium (51.8%).

Table 3 Characteristics of liposomal vinorelbine in combination with a donor of monovalent and multivalent counterions Pharmaceutical composition Types and concentration of donor counterions (mm) Particle size Encapsulation Efficiency % of encapsulated vinorelbine remaining 24 hours after incubation with blood plasma Ammonium sulfate Dextran Sulfate Sodium nm % % LV301 300 2 106,2 101.6 51.8 LV302 300 4 104.5 106.3 67.3 LV303 300 8 101.5 104.6 78.9

Example 5: Effect of various salts of a multivalent counterion donor In vitro study was performed to evaluate the effect of various salts of a multivalent counterion donor on the retention profile of liposomal vinorelbine.

The liposomes prepared according to Example 1 were mixed with 300 mM ammonium sulfate (AS) and two different donor salts of multivalent counterions: the sodium salt of dextran sulfate (DS) and the ammonium salt DS.

The encapsulation efficiency and liposomal vinorelbine retention profile was evaluated in vitro using a plasma-based release method after 24 hours, and the results are summarized in Table 4.

Results: the data show that the sodium salt and dextran sulfate ammonium salt equally contributed to the retention of vinorelbine in the liposome 24 hours after incubation in blood plasma. In addition to this, when the concentration of the multivalent counterion donor or its salt is 10 mM. the retention profile characteristic of the combination of the mono- and multivalent counterion donor (100% and 94.2% of vinorelbine remaining in the liposome after 24 hours) was similar to the retention profile characteristic of the composition of the multivalent counterion donor (98.7% of vinorelbine remaining in the liposome after 24 hours). These data differ from the data shown in Table 3, where when the concentration of the multivalent counterion donor was less than 10 mM, the retention profile of liposomal vinorelbine depended on the concentration of the multivalent counterion donor.

Table 4 Characteristics of liposomal vinorelbine in combination with a donor of monovalent and multivalent counterions Pharmaceutical composition Combination of counterion donors Particle size (nm) Encapsulation Efficiency % of encapsulated vinorelbine remaining 24 hours after incubation with blood plasma LV007 300 mM AS / 10 mM DS 112.7 85.1% 100 LV008 300 mM AS / 10 mM ammonium salt DS 114.1 83.7% 94.2 LV009 10 mm 108.8 89.4% 98.7

ammonium salt DS

Example 6: Effect of Differences in the Molecular Weight of a Multivalent Counterion Donor

The effect of the molecular weight of the multivalent counterion donor on the retention profile of liposomal vinorelbine was evaluated. The liposomes prepared according to Example 1 were mixed with ammonium sulfate and dextran sulfate with a molecular weight of 5K and 8K, respectively.

The encapsulation efficiency and retention profile for various types of liposomal vinorelbine were evaluated in vitro using a plasma release method after 24 hours, and the results are summarized in Table 5.

Results: the total valency of the donor of multivalent counterions affects the retention profile of liposomal vinorelbine. The data indicate that the use of a multivalent counterion donor with a higher valency is associated with an increase in the amount of encapsulated vinorelbine after 24 hours.

Table 5 Characteristics of liposomal vinorelbine used with a donor of multivalent counterions with different molecular weights. Pharmaceutical composition Ammonium sulfate concentration (mm) Dextran sulfate concentration (mM) / molecular weight (K) Particle size (nm) Total valency (meq.) % of encapsulated vinorelbine remaining 24 hours after incubation with blood plasma LV108 300 3 mm / 5K 110.0 73.68 55.9 LV102 300 6 mm / 5K 114.5 147.36 67.7 LV301 300 2 mm / 8K 106,2 78.64 51.8 Pharmaceutical composition Ammonium sulfate concentration (mm) The concentration of dextran sulfate (mm) / molecular Particle size (nm) Total valency (meq.) % encapsulated vinorelbine remaining after 24

mass (K) hours after incubation with blood plasma LV302 300 4 mm / 8K 104.5 157.28 67.3

Example 7: Regulation of the retention profile using a combination of monovalent and multivalent counterion donor

Various pharmaceutical compositions were prepared by mixing the liposomes obtained in Example 1 with various concentrations of ammonium sulfate and various concentrations of dextran sulfate, followed by a remote loading of vinorelbine. The encapsulation efficiency and liposomal vinorelbine retention profile were evaluated in vitro using a plasma-based release method after 24 hours, and the results are summarized in Tables 6-8.

The results are shown in Table 6: after 72 hours, 72.2% of the encapsulated vinorelbine was still in the NanoVNB composition (this pharmaceutical composition contains only the multivalent counterion donor triethylamine octasulfate), and this high retention rate after 72 hours could lead to toxicity. primarily skin toxicity. On the other hand, the entire amount of encapsulated vinorelbine was released from the LV005 composition (this pharmaceutical composition contains only a monovalent counterion donor) after 72 hours, and this is associated with low therapeutic efficacy. By combining donors of monovalent and multivalent counterions, a number of liposomal vinorelbin retention profiles were obtained. It has been noted that the total valency equivalent of a multivalent counterion donor or pharmaceutically acceptable salt is from about 1 to about 240 meq.

Table 6 characteristics of pharmaceutical compositions containing 100 mM and 300 mM monovalent counterion donor and various concentrations of multivalent counterion donor Pharmaceutical composition Dextran sulfate Total valency Ammonium sulfate % of the remaining encapsulated vinorelbine

MW mm meq mm After 24 hours After 72 hours LV005 - - 300 19.8 not detected LV305 8K 0.3 11.79 300 44.1 27.1 LV306 0.6 23.59 Not investigated 31.6 LV301 2 78.62 59.2 44,4 LV304 3 117.93 65.1 Not investigated Pharmaceutical composition Dextran sulfate Total valency Ammonium sulfate % of remaining encapsulated vinorelbine MW mm meq mm After 24 hours After 72 hours LV302 4 157.24 77.1 58.6 LV303 8 314.48 90.1 Unexplored LV402 1,6K 0.25 1.97 47.4 16.5 LV403 0.5 3.93 53.5 20.7 LV404 1 7.86 53.3 24.3 LV40I 1,5 11.79 39 20 Nanovnb 1.2K 75 mM triethylamine octasulfate 600 - 82,2 72,2 LV307 8K 6 235.86 300 49.1 19.6 LV801 8K 4 157.2 100 55.8 15.3 Table 7 characteristics of pharmaceutical compositions containing various concentrations of a monovalent counterion donor and a fixed concentration (0.3 mM) of a multivalent counterion donor

Pharmaceutical composition Types and concentration of donor counterions (mm) Particle size Encapsulation Efficiency Ammonium sulfate Dextran Sulfate Sodium nm % LV801 fifty 0.3 102.3 44,4 LV802 100 0.3 101,2 72.4 LV803 200 0.3 106.9 102.8 LV305 300 0.3 122.1 102.6 LV804 400 0.3 106.1 87.9 LV805 500 0.3 111.2 70,4 LV806 600 0.3 106.5 48.3

The results are shown in Table 7: the encapsulation efficiency of vinorelbine was above 70% when using from 100 mm to 500 mm ammonium sulfate.

Table 8 characteristics of pharmaceutical compositions containing various concentrations of a monovalent counterion donor and a fixed concentration (0.3 mM) of a multivalent counterion donor Pharmaceutical composition Types and concentration of donor counterions (mm) Particle size Encapsulation Efficiency % of encapsulated vinorelbine remaining 24 hours after incubation with blood plasma Ammonium sulfate Dextran Sulfate Sodium nm % % Nanovnb 97.2 100.9 80.3 LV803 200 0.3 106.9 102.8 32,0

LV305 300 0.3 122.1 102.6 44.1 LV804 400 0.3 106.1 87.9 37,4

The results are shown in Table 8: retention of more than 30% liposomal vinorelbine after 24-hour incubation using 200-400 mm ammonium sulfate (donor of monovalent counterions) was observed.

Example 8: evaluation of the anticancer effect in vivo using human colon cancer cells HT-29

The in vivo evaluation of the anticancer effect of the pharmaceutical composition LV304 in an orthotopic tumor model using human NT-29 colon cancer cells in mice was performed.

Mice had free access to drinking water and food throughout this study.

The study plan included 3 study groups, which are listed below:

group receiving NanoNVB: 6 mice were injected once daily with intravenous injection of 25 mg / kg vinorelbine in the form of NanoVNB on days 0, 3, 6 and 9.

the group receiving LV304: 6 mice by intravenous injection once a day were injected with 25 mg / kg of vinorelbine in the form of pharmaceutical composition LV304 on the 0-, 3-, 6- and 9th day.

Control group: 6 mice were given once daily intravenous injection of saline on 0-, 3-, 6- and 9th day.

During the study period, the following indicators were measured:

- percentage change in tumor growth rate (T / C). This indicator was calculated by the following formula:

(tumor mass _{day x -} tumor mass _{day 0} ) _{after therapeutic exposure} / mass swollen _{day x -} tumor mass _{day 0} ) _control × 100%.

- the maximum change in body weight compared to body weight on day 0.

- average tumor volume doubling time (TDT). This indicator is often used to quantify tumor growth and is calculated using the following formula:

(day x - day 0)

- day x is the time required to double the volume of the tumor, compared with the size at the time of determining the stage of the malignant tumor.

- an indicator of skin toxicity was evaluated and differentiated based on the parameters listed in Table 9.

Table 9 Skin toxicity Power Signs and severity of signs Hair loss and swelling around the eyelids Hair loss around the inguinal region 0 Not detected Not detected 1 Insignificant Insignificant 2 Moderate Moderate 3 Strong Strong 4 Very strong Very strong

Result:

Table 10 shows that the percentage change in tumor growth rate (% T / C) on day 8 was similar between the groups receiving NanoVNB and LV304 (-41.0% for NanoVNB and -42.4% for LV304). The mean tumor volume doubling time (mean TDT) was> 78 days in the group receiving NanoVNB, 67.1 days in the group receiving LV304, and 7.6 days in the control group. In addition, mice treated with LV304 showed fewer side effects (less weight loss and lower skin toxicity) compared to mice treated with NanoVNB.

In FIG. Figure 2 shows the average tumor volume in the group treated with NanoVNB, in the group treated with LV304, and the group (control) treated with saline. The results indicate that the average tumor volume in the groups treated with NanoVNB and LV 304 was less than 200 mm during the study period, while the average tumor volume in the control group exceeded 3000 mm ³ on the 40th day.

These results suggest that LV304 is an effective anti-cancer therapeutic agent compared to NanoVNB, with fewer side effects.

Table 10 Evaluation of the anticancer effect of NanoVNB, LV304 and physiological saline in a model using NT-29 human colon cancer cells Treatment group % T / C (day) * Max, change in body weight,% (day) * The indicator of skin toxicity (day) * Average TDT Saline - -10.3 (34) - 7.0 ± 2.4 Nanovnb -41.0 ± 15.4 (8) -20.8 (13) 37 (17) > 78 LV304 -42.4 ± 13.1 (8) -4.7 (13) 20 (15) 67.1 ± 5.4 * number of days after day 0

Example 9: evaluation of the anticancer effect in vivo in an orthotopic tumor model using human lung adenocarcinoma cells PC14PE6 / AS2

An in vivo evaluation of the anticancer effect of the pharmaceutical composition LV304 was carried out in an orthotopic lung tumor model in mice using PC14PE6 / AS2 cells.

The study plan included 3 study groups, which are listed below:

The group receiving NanoVNB: 6 mice were injected with 50% of the maximum tolerated dose (MPD) of NanoVNB (1/2 MPD = 7.5 mg / kg of vinorelbine) as a single intravenous injection on day 0.

The group receiving LV304: 6 mice were injected with 50% MTD of the pharmaceutical composition LV304 (1/2 MTD = 10 mg / kg vinorelbine) as a single intravenous injection on day 0.

Control group: 6 mice received a single intravenous injection of saline on day 0.

During the study period, the following indicators were measured:

- The maximum change in body weight, compared with body weight on day 0.

- Average survival time.

Results: Referring to Table 11, the average survival time for mice was 33.8 days after a single injection of NanoVNB, 34.2 days after a single injection of LV304, and 21.4 days after a single injection of saline. In FIG. Figure 3 shows that the group treated with NanoVNB and the group treated with LV304 had a much longer survival time compared to the group (control) treated with saline (p <0.01).

Table 11 Evaluation of the anticancer effect in the groups receiving NanoVNB, LV304 and saline, in an orthotopic tumor model using human lung adenocarcinoma cells PC14PE6 / AS2. Treatment group Max, change in body weight (day) * Average survival time ± SD (day) Saline -3.6 (18) 21.4 ± 2.0 Nanovnb -12.6 (9) 33.8 ± 6.6 LV304 -17.0 (9) 34.2 ± 4.8 * day after drug administration on day 0

Example 10: assessment of skin toxicity in vivo in a model using SCID mice

An in vivo skin toxicity assessment of the pharmaceutical composition LV304 was performed using BALB / c mice. Mice had free access to drinking water and food throughout this study and were randomized to 3 study groups, which are listed below:

The group receiving NanoNVB: 6 mice by intravenous injection once a day received 7.5 mg / kg of vinorelbine in the form of NanoVNB on day 0 and 9.5 mg / kg on the 3rd and 6th day.

The group receiving LV304: 6 mice by intravenous injection once a day received 7.5 mg / kg of vinorelbine in the form of LV304 on day 0 and 9.5 mg / kg on the 3rd and 6th day.

Control group: 6 mice received intravenous injection of physiological saline once a day on days 0, 3, 6, and 9.

Throughout the study period, skin toxicity was assessed and differentiated based on a point scale shown in Table 9.

Results: in FIG. 4 shows skin toxicity indicators in the group treated with NanoVNB and the group treated with LV304. Skin toxicity in the LV304 group was significantly lower compared to the NanoVNB group during the 60-day study period.

In cases where ranges are used in this document to characterize physical properties, such as molecular weight, or chemical properties, such as chemical formulas, it is intended that all combinations and subcombinations of ranges for specific embodiments are incorporated herein.

The disclosures of each patent, patent application, and publication cited or described herein are hereby incorporated by reference in their entirety.

One skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the present invention, and that such changes and modifications can be made without departing from the spirit of the present invention. Thus, it is intended that the appended claims cover all such equivalent variations that fall within the spirit and scope of the invention.

Claims (27)

1. A pharmaceutical composition for inhibiting the growth of cancer cells, containing at least one liposome containing: a particle forming component selected from the group consisting of a phospholipid and a mixture of at least one phospholipid and cholesterol; from 0.1 mM to 10 mM of a multivalent counterion donor or a pharmaceutically acceptable salt thereof, the multivalent counterion donor being a dextran sulfate ion; from 100 mm to 500 mm donor monovalent counterions, and the donor of monovalent counterions is ammonium sulfate; and amphipathic therapeutic agent or a pharmaceutically acceptable salt thereof. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt of the multivalent counterion donor consists of: (i) a dextran sulfate ion; and (ii) a cation, wherein the dextran sulfate ion forms ion pairs with cations. 3. The pharmaceutical composition according to claim 2, wherein the cation comprises at least one cation selected from the group consisting of calcium ion, magnesium ion, sodium ion, potassium ion, manganese ion and NR ₄ ⁺ , where R is H, alkyl or an aryl residue. 4. The pharmaceutical composition according to claim 3, wherein the cation is ammonium. 5. The pharmaceutical composition according to claim 2, wherein the dextran sulfate ion contains sulfate in an amount of from 1 meq to 320 meq per liter. 6. The pharmaceutical composition according to claim 2, wherein the dextran sulfate ion contains sulfate in an amount of from 1 meq to 250 meq per liter. 7. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt of the multivalent counterion donor is sodium dextran sulfate or ammonium dextran sulfate. 8. The pharmaceutical composition according to claim 1, wherein the dextran sulfate ion has a molecular weight of from 1600 daltons to 8000 daltons. 9. The pharmaceutical composition according to claim 1, wherein the concentration of the monovalent counterion donor is from 150 mM to 450 mM. 10. The pharmaceutical composition according to claim 1, wherein the amphipathic therapeutic agent is a pink vinca alkaloid. 11. The pharmaceutical composition according to claim 1, wherein the amphipathic therapeutic agent is a topoisomerase inhibitor. 12. A pharmaceutical composition for inhibiting the growth of cancer cells, comprising at least one liposome, comprising: a particle forming component selected from a phospholipid or a mixture of at least one phospholipid and cholesterol; 0.1 mM to 10 mM dextran sulfate ion or a pharmaceutically acceptable salt thereof; from 150 mm to 450 mm ammonium sulfate; and pink vinca alkaloid. 13. The pharmaceutical composition of claim 12, wherein the liposome further comprises a hydrophilic polymer. 14. A method of inhibiting the growth of cancer cells, comprising administering an effective amount of a pharmaceutical composition according to any one of claims. 1-13. 15. A liposome for delivering an amphipathic therapeutic agent, comprising: a particle forming component selected from the group consisting of a phospholipid or a mixture of at least one phospholipid and cholesterol; 0.1 mM to 10 mM dextran sulfate ion or a pharmaceutically acceptable salt thereof; and from 150 mm to 450 mm ammonium sulfate. 16. The liposome according to claim 15, wherein the dextran sulfate ion has a molecular weight of from 1600 daltons to 8000 daltons.

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